This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. The goal of this project is to image the mitochondrial redox state of individual embryonic stem cell colonies to investigate the possible association between the redox state and the pluripotency of stem cells to ultimately develop a novel ESC sorter using the mitochondrial redox state as the cell sorting marker. The first step was set to image the redox state of the individual mouse ESC colonies without any disturbance of these colonies and to compare the result with the immunochemistry of the Oct4-stained colonies. The second step was set to transfect the ESC with another embryonic stem cell marker Nanog and simultaneously image the Nanog expression and the mitochondrial redox state. The third step was set to calibrate the redox state with appropriate agents. The fourth step was set to image the ESC colonies at the single cell resolution using a confocal microscope.